Hybrid Duplex Research Articles

Template Misalignment in Multisubunit RNA Polymerases and Transcription Fidelity

Recent work showed that the single-subunit T7 RNA polymerase (RNAP) can generate misincorporation errors by a mechanism that involves misalignment of the DNA template strand. Here, we show that the same mechanism can produce errors during transcription by the multisubunit yeast RNAP II and bacterial RNAPs. Fluorescence spectroscopy reveals a reorganization of the template strand during this process, and molecular modeling suggests an open space above the polymerase active site that could accommodate a misaligned base. Substrate competition assays indicate that template misalignment, not misincorporation, is the preferred mechanism for substitution errors by cellular RNAPs. Misalignment could account for data previously taken as evidence for additional NTP binding sites downstream of the active site. Analysis of the effects of different template topologies on misincorporation indicates that the duplex DNA immediately downstream of the active site plays an important role in transcription fidelity.

Analogues of a Locked Nucleic Acid with Three-Carbon 2‘,4‘-Linkages: Synthesis by Ring-Closing Metathesis and Influence on Nucleic Acid Duplex Stability and Structure

Two bicyclic 2'-deoxynucleoside analogues containing a saturated and an unsaturated three-carbon 2',4'-linkage, respectively, have been synthesized using a ring-closing metathesis-based linear strategy starting from uridine. Both analogues have been incorporated into oligodeoxynucleotide sequences and increased the stability of DNA:RNA hybrid duplexes (DeltaT(m) approximately 2.5-5.0 degrees C per modification) and decreased the stability of dsDNA duplexes (DeltaT(m) approximately 2.5-1.0 degrees C per modification). CD spectroscopy revealed that the bicyclic nucleosides induced formation of A-type-like duplexes albeit to a lesser degree than found for locked nucleic acid (LNA) monomers. From the CD data and UV melting analysis, we propose that the 2'-oxygen atom of the bicyclic moiety is essential for the formation of stabilized A-type-like dsDNA but not for the formation of a stabilized A-type DNA:RNA hybrid.

Antisense locked nucleic acids efficiently suppress BCR/ABL and induce cell growth decline and apoptosis in leukemic cells

Chronic myeloid leukemia (CML) develops when a hematopoietic stem cell acquires the Philadelphia chromosome carrying the BCR/ABL fusion gene. This gives the transformed cells a proliferative advantage over normal hematopoietic cells. Silencing the BCR/ABL oncogene by treatment with specific drugs remains an important therapeutic goal. In this work, we used locked nucleic acid (LNA)-modified oligonucleotides to silence BCR/ABL and reduce CML cell proliferation, as these oligonucleotides are resistant to nucleases and exhibit an exceptional affinity for cognate RNA. The anti-BCR/ABL oligonucleotides were designed as LNA-DNA gapmers, consisting of end blocks of 3/4 LNA monomers and a central DNA stretch of 13/14 deoxyribonucleotides. The gapmers were complementary to the b2a2 and b3a2 mRNA junctions with which they form hybrid duplexes that have melting temperatures of 79 degrees C and 75 degrees C, respectively, in a 20 mmol/L NaCl-buffered (pH 7.4) solution. Like DNA, the designed LNA-DNA gapmers were capable of activating RNase H and promote cleavage of the target b2a2 and b3a2 BCR/ABL mRNAs. The treatment of CML cells with junction-specific antisense gapmers resulted in a strong and specific reduction of the levels of BCR/ABL transcripts ( approximately 20% of control) and protein p210(BCR/ABL) ( approximately 30% of control). Moreover, the antisense oligonucleotides suppressed cell growth up to 40% of control and induced apoptosis, as indicated by the increase of caspase-3/7 activity in the treated cells. Finally, the b2a2-specific antisense gapmer used in combination with STI571 (imatinib mesylate), a tyrosine kinase inhibitor of p210(BCR/ABL), produced an enhanced antiproliferative effect in KYO-1 cells, which compared with K562 cells are refractory to STI571. The data of this study support the application of BCR/ABL antisense LNA-DNA gapmers, used either alone or in combination with STI571, as potential antileukemic agents.

Two-dimensional LNA/DNA arrays: estimating the helicity of LNA/DNA hybrid duplex

We measured the helical repeats of a non-natural nucleic acid, locked nucleic acid (LNA), by incorporating LNA strands into the outer arms of a DNA double crossover (DX) molecule; atomic force microscopy (AFM) imaging of the two-dimensional (2D) arrays self-assembled from these DX molecules allows us to derive the helical repeat of the LNA/DNA hetero-duplex to be 13.2 +/- 0.9 base pairs per turn.

Consequences of P‐Chirality in Chimeric 2′‐O‐Methyloligoribonucleotides with Stereoregular Methylphosphonothioate Linkages

AbstractStereoregular chimeric oligonucleotides modified with diastereomerically pure methylphosphonothioate linkages interact with complementary DNA or RNA templates in a stereodependent manner. There is an unprecedented large difference in the stability of duplexes of oligonucleotides modified with alternating methylphosphonothioate linkages (ΔTm = 34 °C) that is dependent only on the stereochemistry of the methylphosphonothioate phosphorus centres. The nature of these interactions was studied by circular dichroism and NMR spectroscopy, and a thermodynamic analysis was performed. The collected data indicate that the absolute configuration at the P‐chiral methylphosphonothioate linkages in chimeric 2′‐O‐methylribonucleotides could, in some cases, determine the global conformation of single–strand oligonucleotides and constrain the conformation of hybrid duplexes formed with RNA. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

A Ribose Sugar Conformational Switch in the LTR-Retrotransposon Ty3 Polypurine Tract-Containing RNA/DNA Hybrid

To probe structural features of a polypurine tract (PPT) that mediate its specific recognition and processing, a model 20 bp RNA/DNA hybrid duplex, which includes the full PPT sequence of the Saccharomyces cerevisiae LTR-retrotransposon Ty3, has been investigated using solution NMR spectroscopy. While homonuclear NOESY and DQF-COSY analyses indicate that this PPT-containing RNA/DNA hybrid adopts an overall A-form-like helical geometry, an unexpected sugar pucker switch has been detected for the ribose at position +1, relative to the cleavage site, on the RNA strand. A model of the conformational changes induced by the A- to B-type sugar pucker switch shows a significant change in the backbone trajectory of the RNA strand, which alters the presentation of backbone phosphate and 2' hydroxyl groups 3' of this residue. This observation implies that part of the mechanism governing RNase H fidelity may be through distortion of the RNA/DNA helix one base ahead of the scissile bond.

X-Ray analyses of hybrid duplexes between antisense oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2′-O-methyluridine and their target RNAs

Incorporation of 5-(N-aminohexyl)carbamoyl-2'-O-methyluridine ((N)Um) into oligonucleotides increases antisense properties such as RNA binding affinity, nuclease resistance and RNase H activity. The present X-ray studies on hybrid duplexes formed between antisense oligonucleotides containing (N)Um and their target RNAs have revealed the structural basis for such properties. The terminal ammonium groups of the aminohexyl chains interact with the phosphate oxygen anions. The 2'-O-methyl modification induces the ribose group to adopt the C3'-endo conformation. Comparisons with the structure of unmodified duplex show that the (N)Um incorporation narrows the minor grooves and alters their hydration structures. These structural changes are well correlated to the favorable properties for useful antisense molecules.

Base Pair Interactions and Hybridization Isotherms of Matched and Mismatched Oligonucleotide Probes on Microarrays

The microarray technology enables the expression degree of thousands of genes to be estimated at once by the measurement of the abundance of the respective messenger RNA. This method is based on the sequence specific binding of RNA to DNA probes and its detection using fluorescent labels. The raw intensity data are affected by the sequence-specific affinity of probe and RNA for duplex formation, by the background intensity due to nonspecific hybridization at small transcript concentrations and by the saturation of the probes at high transcript concentration owing to surface adsorption. We address these issues using a binding model which describes specific and nonspecific hybridization in terms of a competitive two-species Langmuir isotherm and DNA/RNA duplex formation in terms of sequence-specific, single-base related interactions. The GeneChip microarrays technology uses pairs of so-called perfect match (PM) and mismatch (MM) oligonucleotide probes to estimate the amount of nonspecific hybridization. The mean affinity of the probes decrease according to PM(specific) > MM(specific) >> PM(nonspecific) approximately MM(nonspecific). The stability of specific and nonspecific DNA/RNA duplexes is mainly determined by Watson Crick (WC) pairings. Mismatched self-complementary pairings in the middle of the MM sequence only weakly contribute to the duplex stability. The asymmetry of base pair interaction in the DNA/RNA hybrid duplexes gives rise to a duplet-like symmetry of the PM - MM intensity difference at dominating nonspecific hybridization and a triplet-like symmetry at specific hybridization. The signal intensities of the PM and MM probes and their difference are assessed in terms of sensitivity and specificity. The presented results imply the refinement of existing algorithms of probe level analysis to correct microarray data for nonspecific background intensities and saturation on the basis of the probe sequence.

Physical-layer design for next-generation cellular wireless systems

The conventional cellular architecture will remain an integral part of next-generation wireless systems, providing high-speed packet data services directly to mobile users and also backhaul service for local area networks. In this paper, we present several proposals addressing the challenges associated with the critical design issues for the next-generation cellular wireless physical layer. These proposals include an orthogonal frequency division multiplexed (OFDM) or multicarrier code division multiple access (MC-CDMA) air interface for wide channel bandwidths, considerations for resource allocation, multiple antenna techniques, a hybrid duplexing technique combining the benefits of time division and frequency division duplexing, and coordinated networks for reducing intercell interference and increasing overall spectral efficiency.

Hybrid duplex: a novel method to study the contractile function of heterogeneous myocardium

In an earlier study, we experimentally mimicked the effects of mechanical interaction between different regions of the ventricular wall by allowing pairs of independently maintained cardiac muscle fibers to interact mechanically in series or in parallel. This simple physiological model of heterogeneous myocardium, which has been termed "duplex," has provided new insight into basic effects of cardiac electromechanical heterogeneity. Here, we present a novel "hybrid duplex," where one of the elements is an isolated cardiac muscle and the other a "virtual cardiac muscle." The virtual muscle is represented by a computational model of cardiomyocyte electromechanical activity. We present in detail the computer-based digital control system that governs the mechanical interaction between virtual and biological muscle, the software used for data analysis, and working implementations of the model. Advantages of the hybrid duplex method are discussed, and experimental recordings are presented for illustration and as proof of the principle.

RNA Guanine Quadruplex Invasion by Complementary and Homologous PNA Probes

Guanine quadruplexes are gaining increasing attention due to their suspected roles in regulating gene expression at the transcriptional and translational levels. This paper describes the ability of short peptide nucleic acid (PNA) probes to disrupt a stable RNA quadruplex and hybridize to their target sequence. In one case, the PNA probe is complementary to the target, resulting in formation of a Watson-Crick base-paired duplex. In the second case, the PNA probe is homologous to the target and forms a hybrid quadruplex structure. The hybrid duplex is formed in a 1:1 stoichiometry, as expected based on the constraints imposed by Watson-Crick pairing. However, the hybrid quadruplex is formed in a PNA2:RNA stoichiometry, due to the ability of the short PNA to hybridize with both halves of the original RNA quadruplex.

Structural features of a central mismatch in oligonucleotide hybrid duplexes visualized via Raman spectroscopy: Model system for evaluation of potential “antisense” drugs

Structural features of mismatched base pairs were studied on four nonamer hybrid duplexes formed between the 5'-d(GTGATATGC)-3' complement and its 5'-r(GCAUNUCAC)-3' (N = A, C, G, U) counterparts. This oligonucleotide set is considered a model molecular system for future systematic studies of various modifications of internucleotide linkages with respect to their impact on the structure of mismatched base pairs. Raman spectra, measured at 15 degrees C, revealed the prevailing A-like structure of the RNA strand and mixed A-like and B-like characteristics for the DNA strand. All three mismatches disturb only weakly the overall conformation of the hybrid duplex in contrast to analogous mismatched DNA duplexes. In particular, the dT x rG mismatch influences the global hybrid duplex geometry almost negligibly. The dT x rC and dT x rU mismatches induce somewhat more pronounced distortions of the backbone structure and of the thymine position, the latter being expressed by a change of the surrounding methylene group without effect on the carbonyl's vibrations. Structural effects of the mismatches correlate well with the duplex thermodynamic stabilities obtained by ultraviolet (UV) absorption, i.e., the dT x rG mismatch decreases the hybrid duplex stability very weakly while the effect of both pyrimidine-pyrimidine mismatches is considerable.

Reinforced HNA Backbone Hydration in the Crystal Structure of a Decameric HNA/RNA Hybrid

The crystal structure of a decameric HNA/RNA (HNA = 2',3'-dideoxy-1',5'-anhydro-d-arabinohexitol nucleic acid) hybrid with the RNA sequence 5'-GGCAUUACGG-3' is the first crystal structure of a hybrid duplex between a naturally occurring nucleic acid and a strand, which is fully modified to contain a six-membered ring instead of ribose. The presence of four duplex helices in the asymmetric unit allows for a detailed discussion of hydration, which revealed a tighter spinelike backbone hydration for the HNA- than for the RNA-strands. The reinforced backbone hydration is suggested to contribute significantly to the exceptional stability of HNA-containing duplexes and might be one of the causes for the evolutionary preference for ribose-derived nucleic acids.

Chirality at phosphorus: hybrid duplexes of chimeric oligonucleotides containing methylphosphonothioate linkages with complementary DNA and RNA

Chimeric oligonucleotides with incorporated diastereomerically pure dinucleoside(3′,5′)-methylphosphonothioates and their oxo- and seleno- congeners of known absolute configuration are reported. The relation between stability of the hybrid duplexes with complementary DNA and RNA and their structure is analyzed in context of absolute configuration of the P-chiral internucleotide bonds.

Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor Combinations on HIV-1 Reverse-Transcriptase Activities

Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3'-azido-3'-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.

The 5-Me of thyminyl (T) interaction with the neighboring nucleobases dictate the relative stability of isosequential DNA–RNA hybrid duplexes

Relative energetic contributions from the base-pairing [DeltaG(degrees)(bp)] vis-a-vis stacking [DeltaG(degrees)(stacking)] to the total free-energy of stabilization [DeltaG(degrees)(37)] for 14 pairs of isosequential hybrid DNA-RNA duplexes (taken from E. A. Lesnik and S. M. Freier, Biochemistry ,1995, 34, 10807) have been dissected in order to understand the differences in the intrinsic nature of the electrostatic forces that are responsible for the self-assembly of the heteroduplexes compared to homoduplexes. The pKa differences between the monomeric nucleotide 3'-ethylphosphates [(d/rN)pEt] as well as nucleotide 3',5'-bis-ethylphosphates [Etp(d/rN)pEt] in both 2'-deoxy (dN) and ribo (rN) series (N = A/G/C/T/U), as the model donor and acceptor (in which stacking is completely eliminated) mimicking those of the internucleotide monomer building blocks of a duplex, can be qualitatively used (P. Acharya, P. Cheruku, S. Chatterjee, S. Acharya and J. Chattopadhyaya, J. Am. Chem. Soc., 2004, 126, 2862) to understand the strength of base-pairing energies in different DNA-RNA (DR), RNA-DNA (RD), DNA-DNA (DD), and RNA-RNA (RR) duplexes. The study has led us to show the following. (1) As the number of excess %T in DR duplexes compared to the isosequential RD duplexes increase the differences in their thermal stabilization [DeltaT(m)](DR-RD) increase and vice-versa(2) The total relative stabilizations, [DeltaDeltaG(degrees)(37)](DR-RD) among the 14 pairs of isosequential DR and RD duplexes (E. A. Lesnik and S. M. Freier, Biochemistry ,1995, 34, 10807) are wholly dependent on the differences in the number of 5-Me(T) stacking interactions with the nearest-neighbors in the D strands of DR duplexes compared to that of the RD duplexes (3) In the relative stabilization of the DR or RD duplexes differences in the free-energy of stackings [DeltaDeltaG(degrees)(stacking)](DR-RD) play a more significant role than the differences in the free-energy of base-pairing, [DeltaDeltaG(degrees)(bp)](DR-RD). In contradistinction, our experimental data shows that RNA-RNA duplexes are more stable than DNA-DNA duplexes because of larger energy gain from the base-pairing in the former compared to the latter (P. Acharya, P. Cheruku, S. Chatterjee, S. Acharya and J. Chattopadhyaya, J. Am. Chem. Soc., 2004, 126, 2862).

Site-Selective RNA Cleavage by DNA Bearing a Base Pair-Mimic Nucleoside

We have synthesized the deoxyadenosine derivative tethering a phenyl group (X), which mimics the Watson-Crick A/T base pair. The RNA/DNA hybrid duplexes containing X in the middle of the DNA sequence showed a similar thermal stability regardless of the ribonucleotide species (A, G, C, or U) opposite to X, probably because of the phenyl group stacking inside of the duplex accompanied by the opposite ribonucleotide base flipped in an extrahelical position. The RNA strand hybridized with the DNA strand bearing X was cleaved on the 3'-side of the ribonucleotide opposite to X in the presence of MgCl2, and the RNA sequence to be cleaved was not restricted. The site-specific RNA hydrolysis suggests that the DNA strand bearing X has the advantage of the site-selective base flipping in the target sequence and the development of a "universal deoxyribozyme" to exclusively cleave a target RNA sequence.

Synthesis, physicochemical and biochemical studies of 1',2'-oxetane constrained adenosine and guanosine modified oligonucleotides, and their comparison with those of the corresponding cytidine and thymidine analogues.

We have earlier reported the synthesis and antisense properties of the conformationally constrained oxetane-C and -T containing oligonucleotides, which have shown effective down-regulation of the proto-oncogene c-myb mRNA in the K562 human leukemia cells. Here we report on the straightforward syntheses of the oxetane-A and oxetane-G nucleosides as well as their incorporations into antisense oligonucleotides (AONs), and compare their structural and antisense properties with those of the T and C modified AONs (including the thermostability and RNase H recruitment capability of the AON/RNA hybrid duplex by Michaelis-Menten kinetic analyses, their resistance in the human serum, as well as in the presence of exo and endonucleases).

Assembly of an antiparallel homo-adenine DNA duplex by small-molecule binding.

Molecules that reversibly bind DNA and trigger the formation of non-Watson-Crick secondary structures would be useful in the design of dynamic DNA nanostructures and as potential leads for new therapeutic agents. We demonstrate that coralyne, a small crescent-shaped molecule, promotes the formation of a duplex secondary structure from homo-adenine oligonucleotides. AFM studies reveal that the staggered alignment of homo-adenine oligonucleotides upon coralyne binding produces polymers of micrometers in length, but only 2 nm in height. A DNA duplex was also studied that contained eight A.A mismatches between two flanking 7-bp Watson-Crick helices. CD spectra confirm that the multiple A.A mismatches of this duplex bind coralyne in manner similar to that of homo-adenine oligonucleotides. Furthermore, the melting temperature of this hybrid duplex increases by 13 degrees C upon coralyne binding. These observations illustrate that the helical structure of the homo-adenine-coralyne duplex is compatible with the B-form DNA helix.

Effect of LNA Modifications on Small Molecule Binding to Nucleic Acids

Locked nucleic acid (LNA) is a conformationally constrained DNA analogue that exhibits exceptionally high affinity for complementary DNA and RNA strands. The deoxyribose sugar is modified by a 2′-O, 4′-C oxymethylene bridge, which projects into the minor groove. In addition to changing the distribution of functional groups in the groove and the overall helical geometry relative to unmodified DNA, the bridge likely alters the hydration of the groove. Each of these factors will impact the ability of small molecules, proteins and other nucleic acids to recognize LNA-containing hybrids. This report describes the ability of several DNA-intercalating ligands and one minor groove binder to recognize LNA-DNA and LNA-RNA hybrid duplexes. Using UV-vis, fluorescence and circular dichroism spectroscopies, we find that the minor groove binder as well as the intercalators exhibit significantly lower affinity for LNA-containing duplexes. The lone exception is the alkaloid ellipticine, which intercalates into LNA-DNA and LNA-RNA duplexes with affinities comparable to unmodified DNA-DNA and RNA-DNA duplexes.